Much effort has been expended in recent years to develop methods for measuring activation products of fibrinogen. The reason is that fibrinogen to fibrin conversion may be involved in many different pathological conditions, including the prethrombotic state and disseminated intravascular coagulation. Nevertheless, the presently available methods for direct determination of circulating fibrin (soluble fibrin) are not practical since they are either difficult to perform or suffer lack of sensitivity and of specificity. Such methods have consisted, among others, of chemical purification and chromatographic assay of soluble fibrin in human plasma or by a serial dilution protamine sulfate testing.
In theory, fibrin formation can be detected indirectly by determination of released fibrinopeptides. However, apart from being difficult to analyze, the fibrinopeptides have short half-lives and may not give the same information as direct determination of soluble fibrin.
It has also been proposed to use a method based on the stimulatory effect of fibrin obtained on the t-PA-catalyzed activation of plasminogen (Wiman et al., Thrombosis and Haemostatis 1986, 55:(2) 189). In this assay the t-PA activity increases in the presence of soluble fibrin and can be back-calculated to indicate soluble fibrin concentration. The end point determination, however, is one of enzymatic activity by t-PA on a plasmin-sensitive chromogenic substrate rather than a degradation fragment of the fibrin.
It has also been proposed to use immunoassays that utilize monoclonal antibodies raised against the unique antigenic determinants of crosslinked fibrin to specifically measure the degradation products of crosslinked fibrin without being influenced by the degradation products of fibrinogen (Rylatt et al. Thromb. Res. 1983, 31:767-78) (Whitaker et al., J. Clin. Pathol. 1984; 37:882-7) (Gaffney et al., Thrombosis and Haemostasis 1987; 58:231, abstract). The D-dimer is one of the degradation products of crosslinked fibrin which is stable and resists further digestion by plasmin. Elevated serum or plasma levels of D-dimer have been found in patients with pathologic fibrinolysis and after streptokinase-induced thrombolysis in patients with deep venous thrombosis or pulmonary embolus (Lew et al., J.A.C.C., 1986, Vol. 1, No. 6, 1320-4). Therefore, this technique is potentially useful only for monitoring the end-product of fibrinolysis, for example, during physiologic thrombolysis or with therapy using plasminogen activators.
It would be highly desirable, therefore, to provide a rapid and simple assay that can be used for the in vitro measurement of soluble fibrin concentration, for example, in patient plasma as a useful reflection of the so-called "hypercoagulability state" or pre-thrombotic state reflecting an increased tendency to form fibrin. The assay should be for routine laboratory use and for the determination of large numbers of samples and with great accuracy.
A diagnostic immunoassay for measuring the increased reactivity of a test material for fragment D-dimer produced by the addition of a plasminogen activator to the sample, such as plasma, can be used to determine the original concentration of crosslinked fibrin polymer in the patient sample. Thus, the plasma concentration of D-dimer following degradation of soluble fibrin to D-dimer in vitro could be used as a simple indirect measure of circulating crosslinked fibrin polymer in the in vivo condition.